A major cause of morbidity and mortality among patients with AIDS is cryptococcal meningoencephalitis, caused by infection with the ubiquitous, encapsulated yeast, Cryptococcus neoformans. The natural reservoir of C. neoformans is exogenous, and cryptococcosis is acquired by inhalation of the yeast cells or basidiospores. Although the determinants of virulence have not been elucidated, a growing list of phenotypic properties, including the production of a capsule and a laccase, are known to be essential but not necessarily sufficient for pathogenicity. Indeed, isolates of C. neoformans vary extensively in the expression of many phenotypes, including properties known to affect the clinical outcome. In the previous grant period, DNA-based methods were developed to compare global isolates of C. neoformans from patients with AIDS and other sources, to analyze the distribution and relatedness of strains, and to identify genotypes of clinical importance. In all areas, substantial progress has been made, and this proposal will take advantage of powerful new analytical techniques to continue these studies. Defined genetic markers, such as PCR- RFLP, AFLP, and multigene sequencing, will be used to investigate the population structure of C. neoformans. Specific genotypes will be identified that represent clones that have significantly diverged with respect to clinically relevant phenotypes, including susceptibility to antifungal drugs and the expression of virulence factors. The project will also investigate the relationships between clinical and environmental populations. Population genetic and phylogenetic analyses will be used to estimate patterns of genetic variation, recombination and migration among populations. These approaches will lead to predictive information about the epidemiology, diagnosis and prognosis of cryptococcosis in patients with AIDS.